Drug addiction is a complex disorder with a strong genetic component, while the role of epigenetic factors remains unresolved. We propose that the interplay between genetic polymorphisms and epigenetic changes determines gene expression and possibly mRNA processing, serving a critical role in drug addiction. A large portion of suspected addiction susceptibility genes harbors CpG islands, methylation of which represents a main epigenetic mechanism. Both genetic and epigenetic factors likely contribute to addiction susceptibility and physiological changes occurring as a result of substance abuse. We will study these factors in autopsy tissues from the Miami Brain Endowment Bank, containing ~500 samples from cocaine and other drug abusers and age-matched controls. This repository enables genetic and epigenetic studies in relevant brain regions involved in addiction. CpG methylation can occur randomly between the two allele of a gene (represented in overall expression level), or in an allele-selective fashion. The latter causes an allelic expression imbalance (AEI), which represents a precise and quantitative phenotype for both genetic and epigenetic cis-acting factors. This permits us to address several questions. How does CpG island methylation vary across brain regions, and what is the variability among individuals? Does methylation affect gene expression, alternate promoter usage, or alternative splicing? What is the effect of substance abuse on CpG island methylation in candidate genes, in relevant brain regions? Do epigentic and genetic factors contribute to clinical status (addiction)? In this project, we target genes harboring CpG islands that are implicated in addiction, focusing on biogenic amine pathways, encoding synthetic and catabolic enzymes, vesicular and synaptic reuptake transporters, and receptors (MAOA, MAOB, COMT, TH, DAT, NET, VMAT2, DRD2, CHRNA4). We have developed high-throughput tools for measuring the genetic and epigenetic contribution to mRNA and protein expression, and alternative splicing. Our assays are allele-specific, enabling the evaluation of genetic and epigenetic factors in allelic expression, a powerful tool for assessing the quantitative impact of each factor. This novel approach, applied to anatomically defined brain tissues from drug addicts and controls, has the potential to yield significant insight into the role of and interplay between genetic and epigenetic factors, and add to our understanding of susceptibility to addiction.